The overall goal of this proposal is to understand the mechanisms of thrombin-regulated signaling. Thrombin, a coagulant protease, elicits a variety of cellular effects that are essential for hemostasis and thrombosis, as well as inflammatory and proliferative responses produced by vascular damage. Therefore, understanding the mechanisms of thrombin signaling may provide new strategies for the prevention and treatment of thrombin-related cardiovascular diseases. Protease-activated receptors (PARs) are G protein-coupled receptors (GPCRs) that mediate most of thrombin responses in cells. PAR1, the prototype of this family, is the predominant mediator of thrombin signaling in human platelets, as well as in endothelial, fibroblast and smooth muscle cells. PARI is activated by an unusual proteolytic mechanism, and is then internalized and sorted directly to lysosomes. The mechanisms that contribute to termination of PAR1 signaling are not clear. ?arrestins bind to phosphorylated GPCRs to mediate desensitization and internalization. PAR1 signaling is terminated by phosphorylation raising the possibility that ?arrestins function in this process. ?arrestins function as scaffolds to link GPCRs to ERK1/2 kinase activation, whether ?arrestins facilitate PAR1 signaling to ERK1/2 kinase activation is not known. We will utilize mouse embryonic fibroblasts (MEFs) derived from ?arrestin knockouts to delineate the role of ?arrestins in PAR1 signaling. We previously showed that internalization and lysosomal sorting of activated PAR1 are also critical for termination of receptor signaling. PAR1 is internalized via a clathrin- and dynamin-dependent mechanism that is independent of arrestins. The mechanism by which activated PAR1 is internalized through clathrin-coated pits remains poorly understood. We have recently identified sorting nexin 1 as being involved in lysosomal sorting of PAR1. The mechanism by which SNX1 regulates lysosomal sorting of PAR1 is not known. The specific aims of this proposal are to: (1) delineate the role of ?arrestins in the regulation of PAR1 signaling, (2) determine the contributions of phosphorylation versus ?arrestin binding to PAR1 desensitization, and (3) define the molecular mechanisms by which activated PAR1 is internalized and sorted to lysosomes. The studies outlined in this proposal are directed towards understanding the molecular mechanisms involved in termination of PAR1 signaling. [unreadable] [unreadable]